Abstract

Innate immunity is the first line of defence against pathogens and is essential for survival of the infected host. The fruit fly Drosophila melanogaster is an emerging model to study viral pathogenesis, yet antiviral defence responses remain poorly understood. Here, we describe the heat shock response, a cellular mechanism that prevents proteotoxicity, as a component of the antiviral immune response in Drosophila. Transcriptome analyses of Drosophila S2 cells and adult flies revealed strong induction of the heat shock response upon RNA virus infection. Dynamic induction patterns of heat shock pathway components were characterized in vitro and in vivo following infection with different classes of viruses. The heat shock transcription factor (Hsf), as well as active viral replication, were necessary for the induction of the response. Hsf-deficient adult flies were hypersensitive to virus infection, indicating a role of the heat shock response in antiviral defence. In accordance, transgenic activation of the heat shock response prolonged survival time after infection and enabled long-term control of virus replication to undetectable levels. Together, our results establish the heat shock response as an important constituent of innate antiviral immunity in Drosophila.

Transcriptome analysis after RNA sequencing of DCV or CrPV-infected flies.

(a) Overview of the experimental workflow. Female flies (3-5 days old) were inoculated with DCV, CrPV (TCID50 = 10,000), or Tris buffer (mock infection) and total RNA was extracted at 24 hpi for next-generation sequencing. Figure drawn by S.H. Merkling. (b) Number of differentially expressed genes (≥2-fold over mock) at 24 hpi with DCV or CrPV. (c) Venn diagram representing the overlap between the upregulated genes (≥2-fold over mock) upon DCV and CrPV infection. (d,e) Gene ontology analysis of the genes that are upregulated by (d) DCV or (e) CrPV at 24 hpi. All significantly enriched level 4 GO terms are shown (P < 0.05 in a hypergeometric test with Benjamini & Hochberg correction). (f,g) Enrichment of predicted transcription factor binding sites amongst genes induced ≥2 fold at 24 hpi with DCV (f) or CrPV (g). The 500-bp region upstream of the transcriptional start site was analysed in Pscan, using the Transfac database. All transcription factors that are significantly enriched over the genome-wide mean are shown (P < 0.05 in a z-test).

(a-c) Survival of wild-type flies, Hsf4 mutants, and Hsf4 mutants carrying a single Hsf transgene (Hsf4; Rescue) upon (a) DCV, (b) CrPV, and (c) IIV-6 infection. (d,e) Survival of flies expressing an RNAi-inducing hairpin targeting Hsf in the fat body upon (d) DCV and (e) CrPV infection. The fat body-specific C564 driver line (C564-Gal4) was used to drive expression of the transcription factor Gal4, which binds to the Upstream Activating Sequence to induce expression a short hairpin targeting Hsf (UAS-HsfRNAi). Flies expressing the C564-Gal4 driver, but not the UAS responder, were included as controls. Mock infections (Tris buffer) were performed along all experiments, and no mortality was noticed over the time course analysed. (f,g) Viral titers of wild-type and Hsf4 mutant flies inoculated with (f) DCV or (g) CrPV over time. The dashed line represents the detection limit of the titration. Data represent mean and s.d. of three biological replicates of at least 15 female flies for each genotype (a-e) or mean and s.d. of three independent experiments, each consisting of 3 replicates of at least 5 female flies for each genotype (f,g). Differences in viral titers were assessed on log-transformed data with a Student’s t-test (*P < 0.05). Statistical analyses for survival assays (a-e) are discussed in the main text.

RNAi and inducible immune pathways are functional in Hsf-deficient flies.

(a) Eye phenotype of 5 to 7-day-old flies expressing a hairpin RNA targeting the Drosophila Inhibitor of Apoptosis thread (thRNAi) and Heat shock factor (Hsf RNAi). The genetic background of the Hsf RNAi line was used as a control (Ctrl). No eye phenotype was observed in control and Hsf RNAi flies that do not expressing the thRNAi transgene. Five representative images are shown for each genotype. (b) In vivo RNAi reporter assay. Reporter plasmids encoding Firefly (Fluc) and Renilla (Ren) luciferase were transfected along with Fluc specific dsRNA or non-specific control dsRNA in Hsf 4 and Dcr2-/- mutant flies and their wild-type controls (CnBw and y1w1, respectively). Reporter gene activity was measured in fly lysates at three days after transfection. Fold silencing by Fluc dsRNA relative to control dsRNA was calculated and presented as the percentage of silencing relative to wild-type controls (see for Fluc/Ren ratios of all samples). Bars represent mean and s.d. of three pools of five flies for each genotype. (c) Expression of inducible immune genes at 24 hpi with DCV (TCID50 = 10,000) determined by RT-qPCR in wild-type or Hsf 4 mutant flies. Expression of the gene of interest was normalized to transcript levels of the housekeeping gene Ribosomal Protein 49 and expressed as fold change relative to mock infection (Tris buffer). Data are mean and s.d. of three independent pools of 15 female flies for each genotype. Student’s t-tests were used to compare the difference in expression between wild-type and mutant flies (*P < 0.05).

(a,b) Survival of flies overexpressing (a) Hsp70 or (b) Hsf. The ubiquitous driver line (Actin-Gal4) was used to drive expression of (a) Hsp70 or (b) Hsf. Flies expressing only the Actin-Gal4 driver or the responders UAS-Hsp70 or UAS-Hsf were included as controls. Mock infections (Tris buffer) were performed along all experiments, and no mortality was noticed over the analysed time course. (c) Viral RNA load was measured by RT-qPCR in single flies over time. The experiment was performed simultaneously with the survival of panel (b). Ten flies were analysed for each time point and genotype. DCV RNA levels were normalized to the housekeeping gene Ribosomal Protein 49 and presented as fold change over the viral RNA levels in flies harvested immediately after inoculation. A Wilcoxon rank-sum test was used to compare differences in viral load (**P < 0.01). Data represent mean and s.d. of three biological replicates (a,b) of at least 15 female flies for each genotype.